PROJECT SUMMARY/ABSTRACT Therapeutic reprogramming of a patient's diseased cells represents a potential curative approach to treating skin diseases like epidermolysis bullosa (EB). Currently, an incomplete understanding of the regulation of skin cell lineage commitment is a major limitation to safely manufacturing genetically-corrected induced pluripotent stem cells (iPSCs). Advances in regenerative medicine demonstrate that treatment of iPSCs with skin-specific morphogen signals drives stem cell differentiation into keratinocytes (KCs), mimicking embryonic development. Preliminary data show that morphogen treatment drives KC lineage commitment through increasing expression of epithelial lineage selectors including p63 and AP-2a. It has become evident, however, that this morphogen treatment also induces dynamic chromatin interactions and genome accessibility to establish a framework for long-range gene regulation during KC lineage commitment. The objective of this application is to define the chromatin landscape and the role of early lineage selectors that are induced by the skin-specific morphogens. The central hypothesis is that the morphogens initiate and establish an epithelial-specific genomic landscape through inducing chromatin looping interactions and expression of lineage-specific licensing factors. Locus-specific and high-throughput chromosome conformation assays will be conducted to define the dynamic chromatin architecture of early differentiating KCs. Additionally, deletion of skin cell lineage licensing factors, such as AP-2a, from cultured stem cells, will allow for the elucidation of how these factors mechanistically enforce epithelial fate commitment. Functional analyses including expression profiling, genome accessibility assays, and genome-wide binding experiments will be performed to characterize subsequent changes in epithelial lineage commitment. The results of these assays will reveal the epigenetic complexities within developmental biology that govern early cell fate determination. This contribution will be significant because it will allow for the more effective characterization of stem cells, which will ultimately benefit the field of regenerative medicine.